PROJECT SUMMARY The exact mechanism underlying the onset of Multiple Sclerosis (MS), a disease that affects over 2 million people worldwide and ~ 400,000 in the United States, is unknown although most experts in the field agree that MS involves an abnormal immune-mediated response against the body?s central nervous system (CNS). Specifically, in the CNS, components of the immune system attack myelin, the protein-based substance that surrounds nerve fiber. This attack on myelin results in multiple scar lesions (hence, Multiple Sclerosis) that lead to disease symptoms. Within the immune system, evidence continues to mount that T-cells are the bloodstream components responsible for the demyelination of the nerve fiber. While it is not clear what triggers the T-cells to attack myelin, it is becoming increasingly clear that in vivo, extracellular ATP may be a major determinant in controlling T-cell function and their passage from the bloodstream to the CNS where they participate in the damage to myelin. Innovative analytical tools are needed to investigate the mechanism of T- cell activation, adhesion, and transfer (extravasation) from the bloodstream to the CNS, at the tissue, cellular, and molecular scales. To meet this need, an investigative team consisting of multiple investigators with expertise in fluidic platforms, nanoscale biosensors, and biological samples, proposes a set of specific aims that will prove that the T-cell activation in MS is due to abnormal glucose processing and ATP production and release by the MS red blood cell. We propose that the development of an innovative microfluidic platform with electrospun fibers will create a unique 3D-environment on a controlled in vitro platform for improved monitoring of T-cell activation/adhesion and extravasation across a tissue cultured to a membrane. Next, we will employ classical cell assay methods to establish that the MS red blood cell has unique glucose processing capabilities stemming from an overexpression of the glucose transporter found in the red blood cell (GLUT1). In aim 3, ion channel modified nanopipettes will be developed to perform quantitative, nanoscale determinations on glucose and ATP transport at the single red blood cell level. These nanoscale sensors will confirm that the somewhat ?global? findings in aim 2 (increased glucose transporter and overproduction of ATP) are indeed affecting glucose uptake, and that the uptake is linked to ATP release, thus providing unprecedented metabolic insight on the genesis of inflammatory response. In the final aim of the proposal, we will combine our tools and discoveries from aims 1-3 with pharmaceutical manipulation of red blood cells obtained from MS patients and controls to determine if abnormal glucose transport in the MS red blood cell is the origin of extracellular ATP production and dysregulation of T-cell activation and adhesion. The successful completion of these aims will not only provide insight into factors affecting demyelination in MS, but also provide platform technologies for cellular analyses across multiple fields.